Thrombosis is a general term for diseases caused by the localized accumulation of circulating blood elements within the vasculature that result in vessel occlusion. Conventional antithrombotic drugs can inhibit thrombus growth by targeting coagulation pathways (for example, heparin and warfarin) or platelet-dependent mechanisms (such as aspirin or clopidogrel). Thrombolytic agents (e.g., streptokinase) are used to degrade thrombi in situ to restore blood flow. Despite advances in this field, the search for new strategies continues because existing treatments impair hemostasis, and must be administered at doses that do not achieve maximum efficacy (Gruber and Hanson, Curr. Pharm. Des. 9(28):2367-2374, 2003).
Hemostasis is a vital function that stops bleeding and protects the integrity of blood circulation on both molecular and macroscopic levels. Hemostasis includes a coagulation cascade of sequentially activatable enzymes that is traditionally divided into three parts: (1) an intrinsic pathway, which includes interactions of blood coagulation proteins that lead to the generation of coagulation factor IXa without involvement of coagulation factor VIIa; (2) an extrinsic pathway, which includes interactions of blood coagulation proteins that lead to the generation of coagulation factor Xa and/or IXa without involvement of factor XI; and (3) a common coagulation pathway, including interactions of blood coagulation proteins II, V, VIII, IX and X that lead to the generation of thrombin. Thrombin activates platelets and generates fibrin, both of which are essential building elements of the hemostatic plug that is responsible for sealing the vascular breach. Complete absence of thrombin or platelets causes paralysis of hemostasis and leads to lethal hemorrhage.
The plasmas of placental and marsupial mammals contain factor XI (fXI) (Ponczek et al., J. Thromb. Haemost. 6:1876-1883, 2008), the zymogen of a plasma protease (fXIa) that contributes to fibrin formation and stability through factor IX activation (Furie et al., Hematology: Basic Principles and Practice, 4th ed. New York: Churchill Livingstone 1931, 2005). fXI deficiency causes a variable trauma-induced hemorrhagic disorder in humans and other species (Seligsohn et al., Thromb. Haemost. 98:84-89, 2007; Knowler et al., J. Am. Vet. Med. Assoc. 205:1557-61, 1994; Ghanem et al., J. Vet. Med. Sci. 67:713-715, 2005; Troxel et al., J. Am. Anim. Hosp. Assoc. 38:549-553, 2002). The physiologic mechanism by which fXI is converted to fXIa has been a topic of debate (Pedicord et al., Proc. Natl. Acad. Sci. U.S.A. 104:12855-12860, 2007; Blat & Seiffert, Thromb. Haemost. 99:457-460, 2008). When blood is exposed to a charged surface, the process of contact activation converts factor XII (fXII) to the protease fXIIa, which then activates fXI (Gailani and Broze, Metabolic and Molecular Basis of Inherited Disease, Scriver et al., eds., New York, N.Y.: McGraw-Hill, pages 4433-4453, 2001). The contribution of this reaction to hemostasis is uncertain as fXII deficiency, unlike fXI deficiency, is not associated with abnormal bleeding in any species in which it has been identified (Gailani and Broze, Metabolic and Molecular Basis of Inherited Disease, Scriver et al., eds., New York, N.Y.: McGraw-Hill, pages 4433-4453, 2001). This is a key piece of supporting evidence for hypotheses proposing that fXI is either activated during hemostasis by a protease distinct from fXIIa, or that auxiliary mechanisms for fXI activation can compensate for the absence of fXIIa (Broze et al., Biochemistry 29:7539-7546, 1990; Davie et al., Biochemistry 30:10363-10370, 1991; Renne et al., Expert Rev. Cardiovasc. Ther. 5:733-741, 2007).
In addition to fXIIa, other candidates for fXI activators include α-thrombin (Naito et al., J. Biol. Chem. 266:7353-7358, 1991; Gailani et al., Science 253:909-912, 1991), meizothrombin (von dem Borne et al., Thromb. Haemost. 78:834-839, 1997), and fXIa (autoactivation) (Naito et al., J. Biol. Chem. 266:7353-7358, 1991; Gailani et al., Science 253:909-912, 1991). Thrombin has received much attention in this regard. Work from several laboratories supports a model in which thrombin or another protease generated early in coagulation activates fXI (von dem Borne et al., Thromb. Haemost. 78:834-839, 1997; von dem Borne et al., Blood 86:3035-3042, 1995; von dem Borne et al., J. Clin. Invest. 99:2323-2327, 1997; Cawthern et al., Blood 91:4581-4592, 1998; Keularts et al., Thromb. Haemost. 85:1060-1065, 2001; Oliver et al., Arterioscler. Thromb. Vasc. Biol. 19:170-177, 1999; Wielders et al., Arterioscler. Thromb. Vasc. Biol. 24:1138-1142, 2004), with fXIa then sustaining coagulation. This hypothesis has been challenged by a study that did not find evidence for fXI activation in thrombin or tissue factor (TF) stimulated plasma in the absence of fXII (Pedicord et al., Proc. Natl. Acad. Sci. U.S.A. 104:12855-12860, 2007). This work also showed that the process of collecting and preparing plasma can generate fXIa, giving the false impression in subsequent assays that fXIIa-independent fXI activation has occurred. These observations have been presented in support of a hypothesis, proposed previously by other investigators (Brunnée et al., Blood 81:580-586, 1993), that normal hemostasis in fXII deficiency reflects loss of fXIIa-initiated processes, such as fibrinolysis, that negate the propensity to bleed from simultaneous loss of fXI activation (Pedicord et al., Proc. Natl. Acad. Sci. U.S.A. 104:12855-12860, 2007; Blat et al., Thromb. Haemost. 99:457-460, 2008).
Coagulation factor XII (fXII) has long been considered a potential target of safe antithrombotic therapy. However, no sufficiently potent inhibitor for fXII activity, such as a potent and useful antibody, has yet been identified. A small molecule fXIa inhibitor has been previously described; however, this irreversible enzyme inhibitor blocks both fXIa and plasma kallikrein (Schumacher et al., Eur. J. Pharmacol. 570(1-3):167-174, 2007). Thus, a need exists for a potent, universal and specific inhibitor of fXI.